Tangential flow depth filtration systems and methods of filtration using same

ABSTRACT

The present disclosure relates to hollow fiber tangential flow filters, including hollow fiber tangential flow depth filters, for various applications, including bioprocessing and pharmaceutical applications, systems employing such filters, and methods of filtration using the same.

PRIORITY

This application claims the benefit of priority under 35 USC § 119 toU.S. Provisional Patent Application Ser. No. 62/640,175, filed Mar. 8,2018, and to U.S. Provisional Patent Application Ser. No. 62/676,411,filed May 25, 2018, which is incorporated by reference herein in itsentirety and for all purposes.

FIELD OF THE DISCLOSURE

Embodiments of this disclosure relate generally to process filtrationsystems, and more particularly to systems utilizing tangential flowdepth filters.

BACKGROUND

Filtration is typically performed to separate, clarify, modify and/orconcentrate a fluid solution, mixture or suspension. In thebiotechnology and pharmaceutical industries, filtration is vital for thesuccessful production, processing, and testing of new drugs, diagnosticsand other biological products. For example, in the process ofmanufacturing biologicals, using animal or microbial cell culture,filtration is done for clarification, selective removal andconcentration of certain constituents from the culture media or tomodify the media prior to further processing. Filtration may also beused to enhance productivity by maintaining a culture in perfusion athigh cell concentration.

Tangential flow filtration (also referred to as cross-flow filtration orTFF) systems are widely used in the separation of particulates suspendedin a liquid phase, and have important bioprocessing applications. Incontrast to dead-end filtration systems in which a single fluid feed ispassed through a filter, tangential flow systems are characterized byfluid feeds that flow across a surface of the filter, resulting in theseparation of the feed into two components: a permeate component whichhas passed through the filter and a retentate component which has not.Compared to dead-end systems, TFF systems are less prone to fouling.Fouling of TFF systems may be reduced further by alternating thedirection of the fluid feed across the filtration element as is done inthe XCell™ alternating tangential flow (ATF) technology commercializedby Repligen Corporation (Waltham, Mass.), by backwashing the permeatethrough the filter, and/or by periodic washing.

Modern TFF systems frequently utilize filters comprising one or moretubular filtration elements, such as hollow-fibers or tubular membranes.Where tubular filtration elements are used, they are typically packedtogether within a larger fluid vessel, and are placed in fluidcommunication with a feed at one end and at the other end with a vesselor fluid path for the retentate; the permeate flows through pores in thewalls of the fibers into the spaces between the fibers and within thelarger fluid vessel. Tubular filtration elements provide large anduniform surface areas relative to the feed volumes they can accommodate,and TFF systems utilizing these elements may be scaled easily fromdevelopment to commercial scale. Despite their advantages, TFF systemsfilters may foul when filter flux limits are exceeded, and TFF systemshave finite process capacities. Efforts to increase process capacitiesfor TFF systems are complicated by the relationship between filter fluxand fouling.

SUMMARY

The present disclosure relates to hollow fiber tangential flow filters,including hollow fiber tangential flow depth filters (also referred toas tangential flow depth filters), for various applications, includingbioprocessing and pharmaceutical applications, systems employing suchfilters, and methods of filtration using the same.

In certain aspects, the present disclosure pertains to filtration ofbioreactor fluids. Bioreactor systems provide an environment supportingbiological activity, which results in the build-up of cell metabolites,including metabolic waste, in the bioreactor fluid. The buildup ofmetabolic waste limits cell amplification and/or cell growth within thebioreactor. As a result, known high capacity bioreactor systems requireeither a very large and expensive bioreactor or require filtering ofbioreactor fluids to maintain optimal biological activity.

In various aspects, the present disclosure pertains to hollow fibertangential flow filters, and in particular hollow fiber tangential flowdepth filters, that comprise the following: a housing having aninterior, a fluid inlet, a retentate fluid outlet, a permeate fluidoutlet, and at least one hollow fiber comprising a porous wall, the atleast one hollow fiber having an interior surface, an exterior surface,and a wall thickness ranging from 1 mm to 10 mm, from 2 mm to 7 mm, 1.5mm to 2 mm, 2 mm to 5 mm, or the like, the interior surface forming aninterior lumen having a width ranging from 0.75 mm to 13 mm, from 1 mmto 5 mm, 1 mm to 2 mm, or the like, and extending though the at leastone hollow fiber. The at least one hollow fiber is positioned in thehousing interior, the fluid inlet and the retentate fluid outlet are influid communication with the interior lumen of the at least one hollowfiber, and the permeate fluid outlet is in fluid communication with thehousing interior and the exterior surface of the porous wall.

In some embodiments, the wall has a mean pore size ranging from 0.2 to10 microns.

In some embodiments, which can be used in conjunction with the aboveaspects and embodiments, the at least one hollow fiber comprises aporous wall that is formed from a plurality of filaments that are bondedtogether.

In some embodiments, the filaments are extruded polymer filaments. Forexample, the extruded polymer filaments may be mono-component filaments.As another example, the extruded polymer filaments may be bi-componentfilaments. Bi-component filaments include those that contain apolyolefin and a polyester, for example, having a polyethyleneterephthalate core and a polypropylene coating.

In some embodiments, which can be used in conjunction with the aboveaspects and embodiments, the extruded polymer filaments are melt-blownfilaments.

In some embodiments, which can be used in conjunction with the aboveaspects and embodiments, a plurality of the extruded polymer filamentsare bonded to one another at spaced apart points of contact to definethe porous wall. For example, a plurality of the extruded polymerfilaments may be thermally bonded to one another at spaced apart pointsof contact to define the porous wall, in which case the hollow fiber maybe formed by assembling the extruded polymer filaments into a tubularshape and heating the extruded polymer filaments such that the extrudedpolymer filaments become bonded to one another, among other techniques.

In some embodiments, which can be used in conjunction with any of theabove aspects and embodiments, the hollow fiber tangential flow filtercomprises plurality of the hollow fibers. In these embodiments, thehollow fiber tangential flow filter may further comprising an inletchamber positioned in an interior of the housing and in fluidcommunication with the fluid inlet, and an outlet chamber positioned inthe interior of the housing and in fluid communication with theretentate fluid outlet, wherein the plurality of hollow fibers extendbetween the inlet chamber and the outlet chamber, and wherein the inletchamber and the outlet chamber are in fluid communication with theinterior lumen of each of the hollow fibers.

In various aspects, a hollow fiber tangential flow filter in accordancewith any of the above aspects and embodiments, is used to separate afluid that comprises large size particles and small size particles intoa permeate comprising the small size particles and a retentatecomprising the large size particles.

In various aspects, the present disclosure is directed to a filtrationmethod that comprises introducing a fluid that comprises large sizeparticles and small size particles into the fluid inlet of a hollowfiber tangential flow filter in accordance with any of the above aspectsand embodiments, wherein the fluid is separated into a permeatecomprising the small particles that exits the hollow fiber tangentialflow filter through the permeate fluid outlet and a retentate comprisingthe large particles that exits the hollow fiber tangential flow filterthrough the retentate fluid outlet.

In some embodiments, which can be used in conjunction with the aboveaspects, the large particles may comprise cells, and the small particlesmay comprise one or more of proteins, viruses, virus like particles(VLPs), exosomes, lipids, DNA, and cell metabolites, among otherpossibilities.

In some embodiments, which can be used in conjunction with the aboveaspects, the fluid further comprises intermediate-sized particles thatare trapped in the wall of the at least one hollow fiber. For example,the large particles may comprise cells, the intermediate-sized particlesmay comprise cell debris, and the small particles may comprise one ormore of proteins, viruses, virus like particles (VLPs), exosomes,lipids, DNA, and cell metabolites, among other possibilities.

In some embodiments, which can be used in conjunction with the aboveaspects and embodiments, the large and small particles are of the samecomposition, and the method is used to separate the small particles fromthe large particles. For example, the large and small particles may beselected from ceramic particles, metal particles, liposomal structuresfor drug delivery, biodegradable polymeric particles, and microcapsules,among other possibilities.

In some embodiments, which can be used in conjunction with the aboveaspects and embodiments, the large particles, small particles andintermediate-sized particles are of the same composition and the methodis used to separate the small particles from the large particles and totrap the intermediate-sized particles in the wall of the at least onehollow fiber. As above, the large, small particles andintermediate-sized particles may be selected from ceramic particles,metal particles, liposomal structures for drug delivery, biodegradablepolymeric particles, and microcapsules, among other possibilities.

In various embodiments, which can be used in conjunction with any of theabove aspects and embodiments, the fluid is fluid from a bioreactor andthe retentate flow is circulated back into the bioreactor.

In various embodiments, which can be used in conjunction with any of theabove aspects and embodiments, the fluid may be introduced into thefluid inlet in a pulsed flow. For example, the pulsed flow may be pulsedat a rate ranging from 1 cycle per minute to 1000 cycles per minute,among other possibilities.

In various aspects, the present disclosure is directed to tangentialflow filtering systems that comprise a pumping system and a hollow fibertangential flow filter in accordance with any of the above aspects andembodiments.

In various embodiments, the pumping system of the tangential flowfiltering system is configured to deliver fluid to the fluid inlet ofthe hollow fiber tangential flow filter in a pulsed flow. For example,the pulsed flow may be pulsed at a rate ranging from 1 cycle per minuteto 1000 cycles per minute, among other possibilities.

In various embodiments, which can be used in conjunction with the aboveaspects and embodiments, the pumping system of the hollow fibertangential flow filtering system may comprise a pulsatile pump. Forexample, the pulsatile pump may be peristaltic pump.

In various embodiments, which can be used in conjunction with the aboveaspects and embodiments, the pumping system of the hollow fibertangential flow filtering system may comprise a pump and a flowcontroller that causes the pump to provide the pulsed flow. For example,the flow controller may be positioned at the pump inlet or the pumpoutlet.

In some embodiments, the flow controller comprises an actuator that isconfigured to periodically restrict flow entering and/or exiting thepump thereby providing pulsed flow to the fluid inlet. For example, theactuator may be selected from an electrically controlled actuator, apneumatically controlled actuator, or a hydraulically controlledactuator. For example, the flow controller may comprise a servo valve ora solenoid valve, among many other possibilities.

In various embodiments, which can be used in conjunction with the aboveaspects and embodiments, the pulsatile pump or flow controller of thetangential flow filtering system may be configured to provide a pulsedflow having a flow rate that is pulsed at a rate ranging from 1 cycleper minute to 1000 cycles per minute.

In various aspects, the present disclosure is directed to bioreactorsystems that comprise (a) a bioreactor vessel configured to containbioreactor fluid, the bioreactor vessel having a bioreactor outlet and abioreactor inlet, (b) a hollow fiber tangential flow filtering system inaccordance with any of the above aspects and embodiments, wherein thebioreactor outlet is in fluid communication with the fluid inlet and thebioreactor inlet is in fluid communication with the retentate outlet.

In various embodiments, the pumping system of the of the hollow fibertangential flow filtering system is configured to provide pulsed flow ofbioreactor fluid into the fluid inlet, thereby separating the pulsedflow of bioreactor fluid into a retentate flow which is re-circulatedfrom the retentate outlet and into the bioreactor inlet and a permeateflow which is collected from the permeate fluid outlet either from thetop or bottom of the housing. In certain embodiments, the pulsed flowmay be pulsed at a rate ranging from 1 cycle per minute to 1000 cyclesper minute, among other possibilities.

In various aspects, the present disclosure is directed to bioreactorsystems comprising (a) a bioreactor vessel configured to containbioreactor fluid, the bioreactor vessel having a bioreactor outlet and abioreactor inlet, (b) a tangential flow filtering system comprising apump and a hollow fiber tangential flow filter in accordance with any ofthe above aspects and embodiments, wherein the bioreactor outlet is influid communication with the fluid inlet and the bioreactor inlet is influid communication with the retentate outlet, and (c) a control system.

In various embodiments, the control system is configured to operate thepump such that a first flow of bioreactor fluid is pumped from thebioreactor outlet and into the fluid inlet, thereby separating the firstflow of bioreactor fluid into a retentate flow which is re-circulatedfrom the retentate outlet and into the bioreactor inlet and a permeateflow which is collected from the permeate fluid outlet.

In some embodiments, which can be used in conjunction with the aboveaspects and embodiments, the bioreactor system is configured to pump thefirst flow of bioreactor fluid in a pulsed fashion. For example, thepulsed flow may be pulsed at a rate ranging from 1 cycle per minute to1000 cycles per minute, among other possibilities.

In various embodiments, a hollow fiber tangential flow filter forbioprocessing may include a housing having an interior, a fluid inlet, aretentate fluid outlet, and a permeate fluid outlet. At least onethick-walled hollow fiber may include a porous wall formed from at leastone polymer. The thick-walled hollow fiber may have an average pore sizeand a density. The wall may define a lumen. The at least one hollowfiber may be disposed in the interior such that the fluid inlet and theretentate fluid outlet are in fluid communication with the lumen and thepermeate fluid outlet is in fluid communication with the interior andthe porous wall. The density may be between 51% and 56% of the densityof an equivalent solid volume of the polymer filaments.

In various embodiments, the density may be about 53%. The average poresize may be about 2 μm with a 90% nominal retention. The polymerfilaments may be melt-blown. The polymer filaments may be sintered. Thepolymer filaments may be selected from the group consisting ofpolyolefin, a polyester, and a combination thereof.

In various embodiments, A bioprocessing system may include a bioreactor.A tangential flow depth filtration (TFDF) unit may include athick-walled hollow fiber formed from at least one polymer and mayinclude a porous wall having a pore size and a density. The porous wallmay define a lumen that is in fluid communication with the bioreactor. Apermeate fluid outlet may be in fluid communication with the porouswall. A pump may be in fluid communication with the lumen. The densitymay be between 51% and 56% of the density of an equivalent solid volumeof the polymer filaments.

In various embodiments, the average pore size may be about 2 μm with a90% nominal retention. The density may be about 53%. The polymerfilaments may be melt-blown. The polymer filaments may be sintered. Thepump may be configured to provide a pulsed flow of fluid through thelumen.

In various embodiments, a method of culturing cells in a perfusionbioreactor system may include a culture vessel fluidly connected to atangential flow depth filtration (TFDF) unit having a retentate channeland a filtrate channel. A culture medium may be flowed from the culturevessel through the retentate channel of the TFDF unit, whereby afraction of the culture medium passes into the filtrate channel. A fluidmay be returned from the retentate channel to the culture vessel. Theculture medium may include at least 60×106 cells/mL. The method may beperformed for at least 8 consecutive days. At least 80% of a pluralityof cells of the culture medium may be viable throughout the 8consecutive days. A volume of fresh culture medium may be added to thesystem that is equal to a permeate volume. Adding the volume of freshculture medium may include adding at least 2 times a volume of theculture vessel to the system per day. The culture medium may include abioproduct of interest. A rate of sieving of the bioproduct of interestmay be at least 99% throughout the 8 consecutive days. The TFDF unit mayinclude a thick-walled hollow fiber that may include melt-blown polymerfilaments. A density of the thick-walled hollow fiber may be between 51%and 56% of the density of an equivalent solid volume of the polymerfilaments. The density may be about 53%. The polymer filaments may beselected from the group consisting of polyolefin, a polyester, and acombination thereof.

In various embodiments, a method of processing a fluid comprising abioproduct may include flowing a culture medium from a process vesselthrough a retentate channel of a TFDF unit. A fraction of the culturemedium may pass into a filtrate channel. A fluid may be returned fromthe retentate channel to the process vessel. The filtrate channel mayinclude a filter having a 2 mm internal diameter lumen therethrough. Thefilter may have an average pore size of about 2 μm. Flowing the culturemedium may be performed at a shear rate of about 8000 s−1. The filtermay have a flux above about 40 L·m−2·hr−1. The filter may have a flux ofabout 2300 L·m−2·hr−1. The flowing step may include the use of a pumpselected from the group consisting of a centrifugal levitating magneticpump, a positive displacement pump, a peristaltic, a membrane pump, andan ATF pump.

In various embodiments, a method of harvesting a bio material from abioreactor system may include a process vessel fluidly connected to atangential flow depth filtration (TFDF) unit having a feed/retentatechannel and a filtrate channel. The method may include flowing a culturemedium via a pump from the process vessel through the feed/retentatechannel of the TFDF unit. A fraction of the culture medium may pass intothe filtrate channel. The fluid may be returned from the feed/retentatechannel to the process vessel. Fluid may be collected from the filtratechannel. The TFDF unit may include a thick-walled hollow fiber formedfrom at least one polymer and may include a porous wall. Thethick-walled hollow fiber may have a density of about 53% of the densityof an equivalent solid volume of the at least one polymer. The porouswall may define a lumen that is in fluid communication with thefeed/retentate channel. The TFDF unit may have a flux above about 400L·m−2·hr−1. The TFDF unit may have a peak cell passage of under 5%. Theculture medium may include a bioproduct of interest. A rate of sievingof the bioproduct of interest may be at least 99%.

In various embodiments, the flowing step may include the use of a pumpselected from the group consisting of a centrifugal levitating magneticpump, a positive displacement pump, a peristaltic, a membrane pump, andan ATF pump.

In various embodiments, a method of harvesting a bio material from abioreactor system may include a process vessel fluidly connected to atangential flow depth filtration (TFDF) unit that may have afeed/retentate channel and a filtrate channel. A culture medium may beflowed via a pump from the process vessel through the feed/retentatechannel of the TFDF unit. A fraction of the culture medium may pass intothe filtrate channel. Fluid may be returned from the feed/retentatechannel to the process vessel. Fluid may be collected from the filtratechannel. The TFDF unit may include a thick-walled hollow fiber formedfrom at least one polymer and may include a porous wall. Thethick-walled hollow fiber may have a density of about 53% of the densityof an equivalent solid volume of the at least one polymer. The porouswall may define a lumen that is in fluid communication with thefeed/retentate channel. The TFDF unit may have a flux above about 400L·m−2·hr−1. The TFDF unit may have a peak cell passage of under 5%. Theculture medium may include a bioproduct of interest. A rate of sievingof the bioproduct of interest may be at least 99%.

In various embodiments, a method of harvesting a bio material from abioreactor system may include a process vessel fluidly connected to atangential flow depth filtration (TFDF) unit that may have afeed/retentate channel and a filtrate channel. A culture medium may beflowed through the feed/retentate channel of the TFDF unit. A fractionof the culture medium may pass into the filtrate channel. The fluid maybe returned from the feed/retentate channel to the process vessel. Thefluid may be collected from the filtrate channel. The TFDF unit mayinclude a thick-walled hollow fiber formed from at least one polymer andmay include a porous wall. The thick-walled hollow fiber may have adensity of about 53% of the density of an equivalent solid volume of theat least one polymer. The porous wall may define a lumen that is influid communication with the feed/retentate channel.

In various embodiments, a pore size of the porous wall may be about 2 μmwith a 90% nominal retention. The flowing step may include the use of apump selected from the group consisting of a centrifugal levitatingmagnetic pump, a positive displacement pump, a peristaltic, a membranepump, and an ATF pump.

BRIEF DESCRIPTION THE DRAWINGS

The above and other aspects of the present disclosure will be moreapparent from the following detailed description, presented inconjunction with the following drawings wherein:

FIG. 1A is a schematic cross-sectional view of a hollow fiber tangentialflow depth filter according to the present disclosure;

FIG. 1B is a schematic partial cross-sectional view of three hollowfibers within a tangential flow filter like that shown in FIG. 1A.

FIG. 2 is a schematic cross-sectional view of a wall of a hollow fiberwithin a tangential flow depth filter like that shown in FIG. 1A.

FIG. 3 is a schematic illustration of a bioreactor system according tothe present disclosure.

FIG. 4A is a schematic illustration of a disposable portion of atangential flow filtering system according to the present disclosure.

FIG. 4B is a schematic illustration of a reusable control systemaccording to the present disclosure.

FIGS. 5A and 5B show normalized permeate pressure versus time forvarious tangential flow filtering systems according to the presentdisclosure.

FIG. 6 shows viable cell density (VCD) and percent viability over timefor perfusion filters, according to an embodiment of the presentdisclosure.

FIG. 7 shows various metrics of a filter of FIG. 6.

FIG. 8 shows a cell growth profile of a filter of FIGS. 6 and 7.

FIG. 9 shows an average percent of sieving for a filter of FIGS. 6-8.

FIG. 10 shows a percent of cells passing through a filter of FIGS. 6-9.

FIG. 11 shows a flux of a filter of FIGS. 6-10.

FIG. 12 shows a turbidity of a filter of FIGS. 6-11.

FIG. 13 shows an empirical comparison of transmembrane pressure changeand filter flux for two TFDF systems of the present disclosure.

DETAILED DESCRIPTION Overview

The embodiments of this disclosure relate, generally, to TFDF, and insome cases to TFDF systems and methods for use in bioprocessing,particularly in perfusion culture and harvest. One exemplarybioprocessing arrangement compatible with the embodiments of thisdisclosure includes a process vessel, such as a vessel for culturingcells (e.g., a bioreactor) that produce a desired biological product.This process vessel is fluidly coupled to a TFDF filter housing intowhich a TFDF filter element is positioned, dividing the housing into atleast a first feed/retentate channel and a second permeate or filtratechannel. Fluid flows from the process vessel into the TFDF filterhousing are typically driven by a pump, e.g., a mag-lev, peristaltic ordiaphragm/piston pump, which may impel fluid in a single direction ormay cyclically alternate the direction of flow.

Bioprocessing systems designed to harvest a biological product at theconclusion of a cell culture period generally utilize a large-scaleseparation device such as a depth filter or a centrifuge in order toremove cultured cells from a fluid (e.g., a culture medium) containingthe desired biological product. These large scale devices are chosen inorder to capture large quantities of particulate material, includingaggregated cells, cellular debris, etc. However, the trend in recentyears has been to utilize disposable or single-use equipment inbioprocessing suites to reduce the risks of contamination or damage thatthat accompanies sterilization of equipment between operations, and thecosts of replacing large scale separation devices after each use wouldbe prohibitive.

Additionally, industry trends indicate that bioprocessing operations arebeing extended or even made continuous. Such operations may extend intodays, weeks, or months of operation. Many typical components, such asfilters, are unable to adequately perform for such lengths of timewithout fouling or otherwise needing maintenance or replacement.

Additionally, in bioprocessing it is often desirable to increase processyields by increasing cell density. However, increasing cell density inmay be complicated by increased filter fouling, etc.

Embodiments of this disclosure address these challenges by providingeconomical filtration means that are tolerant of increased celldensities, extended process times, and suitable for use in harvest. Theinventors have discovered that tangential flow depth filters made bymelt blowing of polymers or polymer blends can be manufactured at acomparatively low-cost compatible with single use, yet are able tooperate for extended periods, at high fluxes, and at increased celldensities.

Exemplary Embodiments

A schematic cross-sectional view of a hollow fiber tangential flowfilter 30 in accordance with present disclosure is shown in FIG. 1A. Thehollow fiber tangential flow filter 30 includes parallel hollow fibers60 extending between an inlet chamber 30 a and an outlet chamber 30 b. Afluid inlet port 32 a provides a flow 12 to the inlet chamber 30 a and aretentate fluid outlet port 32 d receives a retentate flow 16 from theoutlet chamber 30 b. The hollow fibers 60 receive the flow 12 throughthe inlet chamber 30 a. The flow 12 is introduced into a hollow fiberinterior 60 a of each of the hollow fibers 60, and a permeate flow 24passes through walls 70 of the hollow fibers 60 into a permeate chamber61 within a filter housing 31. The permeate flow 24 travels to permeatefluid outlet ports 32 b and 32 c. Although two permeate fluid outletports 32 b and 32 c are employed to remove permeate flow 24 in FIG. 1A,in other embodiments, only a single permeate fluid outlet port may beemployed. Filtered retentate flow 16 moves from the hollow fibers 60into the outlet chamber 30 b and is released from the hollow fibertangential flow filter 30 through retentate fluid outlet port 32 d.

FIG. 1B is a schematic partial cross-sectional view of three hollowfibers 60 within a hollow fiber tangential flow filter analogous to thatshown in FIG. 1A, and shows the separation of an inlet flow 12 (alsoreferred to as a feed) which contains large particles 74 and smallparticles 72 a into a permeate flow 24 containing a portion of the smallparticles and a retentate flow 16 containing the large particles 74 anda portion of the small particles 72 a that does not pass through thewalls 70 of the follow fibers 60.

Tangential flow filters in accordance with the present disclosureinclude tangential flow filters having pore sizes and depths that aresuitable for excluding large particles (e.g., cells, micro-carriers, orother large particles), trapping intermediate-sized particles (e.g.,cell debris, or other intermediate-sized particles), and allowing smallparticles (e.g., soluble and insoluble cell metabolites and otherproducts produced by cells including expressed proteins, viruses, viruslike particles (VLPs), exosomes, lipids, DNA, or other small particles).As used herein a “microcarrier” is a particulate support allowing forthe growth of adherent cells in bioreactors.

In this regard, FIG. 2 is a schematic cross-sectional illustration of awall 70 of a hollow fiber 60 used in conjunction with a hollow fibertangential flow filter 30 like that of FIG. 1A. In FIG. 2, a flow 12comprising large particles 74, small particles 72 a, andintermediate-sized particles 72 b is introduced into the fluid inletport 32 a of the hollow fiber tangential flow filter 30. The largeparticles 74 pass along the inner surface of the wall 70 that forms thehollow fiber interior 60 a (also referred to herein as the fiber lumen)of the hollow fibers and are ultimately released in the retentate flow.The wall 70 includes tortuous paths 71 that capture certain elements(i.e., intermediate-sized particles 72 b) of the flow 12 as a portion ofthe flow 12 passes through the wall 70 of hollow fiber tangential flowfilter 30 while allowing other particles (i e, small particles 72 a) topass through the wall 70 as part of the permeate flow 24. In theschematic cross-sectional illustration of FIG. 2, settling zones 73 andnarrowing channels 75 are illustrated as capturing intermediate-sizeparticles 72 b which enter the tortuous paths 71, while allowing smallerparticles 72 a to pass through the wall 70, thus trappingintermediate-size particles 72 b and causing a separation of theintermediate-size particles 72 b from smaller particles 72 a in thepermeate flow 24. This method is thus different from filtering obtainedby the surface of standard thin wall hollow fiber tangential flow filtermembranes, wherein intermediate-size particles 72 b can build up at theinner surface of the wall 70, clogging entrances to the tortuous paths71.

In this regard, one of the most problematic areas for various filtrationprocesses, including filtration of cell culture fluids such as thosefiltered in perfusion and harvest of cell culture fluids, is decreasedmass transfer of target molecules or particles due to filter fouling.The present disclosure overcomes many of these hurdles by combining theadvantages of tangential flow filtration with the advantages of depthfiltration. As in standard thin wall hollow fiber filters usingtangential flow filtration, cells are pumped through the lumens of thehollow fibers, sweeping them along the surface of the inner surface ofthe hollow fibers, allowing them to be recycled for further production.However, instead of the protein and cell debris forming a fouling gellayer at the inner surface of the hollow fibers, the wall adds what isreferred to herein as a “depth filtration” feature that traps the celldebris inside the wall structure, enabling increased volumetricthroughput while maintaining close to 100% passage of typical targetproteins in various embodiments of the disclosure. Such filters may bereferred to herein as tangential flow depth filters.

As illustrated schematically in FIG. 2, tangential flow depth filters inaccordance with various embodiments of the present disclosure do nothave a precisely defined pore structure. Particles that are larger thanthe “pore size” of the filter will be stopped at the surface of thefilter. A significant quantity of intermediate-sized particles, on theother hand, enter the wall for the filter, and are entrapped within thewall before emerging from the opposing surface of the wall. Smallerparticles and soluble materials can pass though the filter material inthe permeate flow. Being of thicker construction and higher porositythan many other filters in the art, the filters can exhibit enhancedflow rates and what is known in the filtration art as “dirt loadingcapacity,” which is the quantity of particulate matter a filter can trapand hold before a maximum allowable back pressure is reached.

Despite a lack of a precisely defined pore structure, the pore size of agiven filter can be objectively determined via a widely used method ofpore size detection known as the “bubble point test.” The bubble pointtest is based on the fact that, for a given fluid and pore size, withconstant wetting, the pressure required to force an air bubble through apore is inversely proportional to the pore diameter. In practice, thismeans that the largest pore size of a filter can be established bywetting the filter material with a fluid and measuring the pressure atwhich a continuous stream of bubbles is first seen downstream of thewetted filter under gas pressure. The point at which a first stream ofbubbles emerges from the filter material is a reflection of the largestpore(s) in the filter material, with the relationship between pressureand pore size being based on Poiseuille's law which can be simplified toP=K/d, where P is the gas pressure at the time of emergence of thestream of bubbles, K is an empirical constant dependent on the filtermaterial, and d is pore diameter. In this regard, pore sizes determinedexperimentally herein are measured using a POROLUX™ 1000 Porometer(Porometer NV, Belgium), based on a pressure scan method (whereincreasing pressure and the resulting gas flow are measured continuouslyduring a test), which provides data that can be used to obtaininformation on the first bubble point size (FBP), mean flow pore size(MFP) (also referred to herein as “mean pore size”), and smallest poresize (SP). These parameters are well known in the capillary flowporometry art.

In various embodiments, hollow fibers for use in the present disclosuremay have, for example, a mean pore size ranging from 0.1 microns (μm) orless to 30 microns or more, typically ranging from 0.2 to 5 microns,among other possible values.

In various embodiments, the hollow fibers for use in the presentdisclosure may have, for example, a wall thickness ranging from 1 mm to10 mm, typically ranging from 2 mm to 7 mm, more typically about 5.0 mm,among other values.

In various embodiments, hollow fibers for use in the present disclosuremay have, for example, an inside diameter (i.e., a lumen diameter)ranging from 0.75 mm to 13 mm, ranging from 1 mm to 5 mm, 0.75 mm to 5mm, 4.6 mm, among other values. In general, a decrease in insidediameter will result in an increase in shear rate. Without wishing to bebound by theory, it is believed that an increase in shear rate willenhance flushing of cells and cell debris from the walls of the hollowfibers.

Hollow fibers for use in the present disclosure may have a wide range oflengths. In some embodiments, the hollow fibers may have a lengthranging, for example, from 200 mm to 2000 mm in length, among othervalues.

The hollow fibers for use in the present disclosure may be formed from avariety of materials using a variety of processes.

For example, hollow fibers may be formed by assembling numerousparticles, filaments, or a combination of particles and filaments into atubular shape. The pore size and distribution of hollow fibers formedfrom particles and/or filaments will depend on the size and distributionof the particles and/or filaments that are assembled to form the hollowfibers. The pore size and distribution of hollow fibers formed fromfilaments will also depend on the density of the filaments that areassembled to form the hollow fibers. For example, mean pore sizesranging from 0.5 microns to 50 microns may be created by varyingfilament density.

Suitable particles and/or filaments for use in the present disclosureinclude both inorganic and organic particles and/or filaments. In someembodiments, the particles and/or filaments may be mono-componentparticles and/or mono-component filaments. In some embodiments, theparticles and/or filaments may be multi-component (e.g., bi-component,tri-component, etc.) particles and/or filaments. For example,bi-component particles and/or filaments having a core formed of a firstcomponent and a coating or sheath formed of a second component, may beemployed, among many other possibilities.

In various embodiments, the particles and/or filaments may be made frompolymers. For example, the particles and/or filaments may be polymericmono-component particles and/or filaments formed from a single polymer,or they may be polymeric multi-component (i.e., bi-component,tri-component, etc.) particles and/or filaments formed from two, three,or more polymers. A variety of polymers may be used to formmono-component and multi-component particles and/or filaments includingpolyolefins such as polyethylene and polypropylene, polyesters such aspolyethylene terephthalate and polybutylene terephthalate, polyamidessuch as nylon 6 or nylon 66, fluoropolymers such as polyvinylidenefluoride (PVDF) and polytetrafluoroethylene (PTFE), among others.

In various embodiments, a porous wall of a filter may have a densitythat is a percentage of volume that the filaments take up compared to anequivalent solid volume of the polymer. For example, a percent densitymay be calculated by dividing the mass of the porous wall of the filterby the volume that the porous wall takes up and comparing the result, inratio form, to the mass of a non-porous wall of the filament materialdivided by the same volume. A filter having a specific densitypercentage may be produced during manufacturing that has a directrelation to the amount of variable cell density (VCD) at which thefilter can operate without fouling. A density of a porous wall of afilter may additionally or alternatively be expressed by a mass pervolume (e.g., grams/cm3).

Particles may be formed into tubular shapes by using, for example,tubular molds. Once formed in a tubular shape, particles may be bondedtogether using any suitable process. For instance, particles may bebonded together by heating the particles to a point where the particlespartially melt and become bonded together at various contact points (aprocess known as sintering), optionally, while also compressing theparticles. As another example, the particles may be bonded together byusing a suitable adhesive to bond the particles to one another atvarious contact points, optionally, while also compressing theparticles. For example, a hollow fiber having a wall analogous to thewall 70 that is shown schematically in FIG. 2 may be formed byassembling numerous irregular particles into a tubular shape and bondingthe particles together by heating the particles while compressing theparticles.

Filament-based fabrication techniques that can be used to form tubularshapes include, for example, simultaneous extrusion (e.g.,melt-extrusion, solvent-based extrusion, etc.) from multiple extrusiondies, or electrospinning or electrospraying onto a rod-shaped substrate(which is subsequently removed), among others.

Filaments may be bonded together using any suitable process. Forinstance, filaments may be bonded together by heating the filaments to apoint where the filaments partially melt and become bonded together atvarious contact points, optionally, while also compressing thefilaments. As another example, filaments may be bonded together by usinga suitable adhesive to bond the filaments to one another at variouscontact points, optionally while also compressing the filaments.

In particular embodiments, numerous fine extruded filaments may bebonded together to at various points to form a hollow fiber, forexample, by forming a tubular shape from the extruded filaments andheating the filaments to bond the filaments together, among otherpossibilities.

In some instances, the extruded filaments may be melt-blown filaments.As used herein, the term “melt-blown” refers to the use of a gas streamat an exit of a filament extrusion die to attenuate or thin out thefilaments while they are in their molten state. Melt-blown filaments aredescribed, for example, in U.S. Pat. No. 5,607,766 to Berger. In variousembodiments, mono- or bi-component filaments are attenuated as they exitan extrusion die using known melt-blowing techniques to produce acollection of filaments. The collection of filaments may then be bondedtogether in the form of a hollow fiber.

In certain beneficial embodiments, hollow fibers may be formed bycombining bicomponent filaments having a sheath of first material whichis bondable at a lower temperature than the melting point of the corematerial. For example, hollow fibers may be formed by combiningbicomponent extrusion technology with melt-blown attenuation to producea web of entangled biocomponent filaments, and then shaping and heatingthe web (e.g., in an oven or using a heated fluid such as steam orheated air) to bond the filaments at their points of contact. An exampleof a sheath-core melt-blown die is schematically illustrated in U.S.Pat. No. 5,607,766 in which a molten sheath-forming polymer and a moltencore-forming polymer are fed into the die and extruded from the same.The molten bicomponent sheath-core filaments are extruded into a highvelocity air stream, which attenuates the filaments, enabling theproduction of fine bicomponent filaments. U.S. Pat. No. 3,095,343 toBerger shows an apparatus for gathering and heat-treating amulti-filament web to form a continuous tubular body (e.g., a hollowfiber) of filaments randomly oriented primarily in a longitudinaldirection, in which the body of filaments are, as a whole,longitudinally aligned and are, in the aggregate, in a parallelorientation, but which have short portions running more or less atrandom in non-parallel diverging and converging directions. In this way,a web of sheath-core bicomponent filaments may be pulled into a confinedarea (e.g., using a tapered nozzle having a central passageway formingmember) where it is gathered into tubular rod shape and heated (orotherwise cured) to bond the filaments.

In certain embodiments, as-formed hollow fiber may be further coatedwith a suitable coating material (e.g., PVDF) either on the inside oroutside of the fiber, which coating process may also act to reduce thepore size of the hollow fiber, if desired.

Hollow fibers such as those described above may be used to constructtangential flow filters for bioprocessing and pharmaceuticalapplications. Examples of bioprocessing applications in which suchtangential flow filters may be employed include those where cell culturefluid is processed to separating cells from smaller particles such asproteins, viruses, virus like particles (VLPs), exosomes, lipids, DNAand other metabolites.

Such applications include perfusion applications in which smallerparticles are continuously removed from cell culture medium as apermeate fluid while cells are retained in a retentate fluid returned toa bioreactor (and in which equivalent volumes of media are typicallysimultaneously added to the bioreactor to maintain overall reactorvolume). Such applications further include clarification or harvestapplications in which smaller particles (typically biological products)are more rapidly removed from cell culture medium as a permeate fluid.

Hollow fibers such as those described above may be used to constructtangential flow depth filters for particle fractionation, concentrationand washing. Examples of applications in which such tangential flowfilters may be employed include the removal of small particles fromlarger particles using such tangential flow depth filters, theconcentration of microparticles using such tangential flow depth filtersand washing microparticles using such tangential flow filters.

A specific example of a bioreactor system 10 for use in conjunction withthe present disclosure will now be described. With reference to FIGS. 3,4A and 4B, the bioreactor system 10 includes a bioreactor vessel 11containing bioreactor fluid 13, a tangential flow filtering system 14,and a control system 20. The tangential flow filtering system 14 isconnected between a bioreactor outlet 11 a and bioreactor inlet 11 b toreceive bioreactor fluid 12 (also referred to as a bioreactor feed),which contains, for example, cells, cell debris, cell metabolitesincluding waste metabolites, expressed proteins, etc., throughbioreactor tubing 15 from the bioreactor 11 and to return a filteredflow 16 (also referred to as a retentate flow or bioreactor return)through return tubing 17 to the bioreactor 11. The bioreactor system 10cycles bioreactor fluid through the tangential flow filtering system 14which removes various materials (e.g., cell debris, soluble andinsoluble cell metabolites and other products produced by cellsincluding expressed proteins, viruses, virus like particles (VLPs),exosomes, lipids, DNA, or other small particles) from the bioreactorfluid and returns cells to allow the reaction in the bioreactor vessel11 to continue. Removing waste metabolites allows the continuedproliferation of cells within the bioreactor, thereby allowing the cellsto continue to express recombinant proteins, antibodies or otherbiological materials that are of interest.

The bioreactor tubing 15 may be connected, for example, to the lowestpoint or dip tube of the bioreactor 11 and the return tubing 17 may beconnected to the bioreactor 11, for example, in the upper portion of thebioreactor volume and submerged in the bioreactor fluid 13.

The bioreactor system 10 includes an assembly comprising a hollow fibertangential flow filter 30 (described in more detail above), a pump 26,and associated fittings and connections. Any suitable pump may be usedin conjunction with the present disclosure including, for example,peristaltic pumps, positive displacement pumps, and pumps withlevitating rotors inside the pumpheads, among others. As a specificexample, the pump 26 may include a low shear, gamma-radiation stable,disposable, levitating pumphead 26 a, for example, a model numberPURALEV® 200SU low shear re-circulation pump manufactured by Levitronix,Waltham, Mass., USA. The PURALEV® 200SU includes a magneticallylevitated rotor inside a disposable pumphead, and stator windings in thepump body, allowing simple removal and replacement of the pumphead 26 a.

The flow of bioreactor fluid 12 passes from the bioreactor vessel 11 tothe tangential flow filtering system 14 and the return flow of thebioreactor fluid 16 passes from the tangential flow filtering system 14back to the bioreactor vessel 11. A permeate flow 24 (e.g., containingsoluble and insoluble cell metabolites and other products produced bycells including expressed proteins, viruses, virus like particles(VLPs), exosomes, lipids, DNA, or other small particles) is strippedfrom the flow of bioreactor material 12 by the tangential flow filteringsystem 14 and carried away from the tangential flow filtering system 14by tubing 19. The permeate flow 24 is drawn from the hollow fibertangential flow system 14 by a permeate pump 22 into a storage container23.

In the embodiment shown, the tangential flow filtering system 14 (seeFIG. 4A) includes a disposable pumphead 26 a, which simplifies initialset up and maintenance. The pumphead 26 a circulates the bioreactorfluid 12 through the hollow fiber tangential flow filter 30 and back tothe bioreactor vessel 11. A non-invasive transmembrane pressure controlvalve 34 may be provided in line with the flow 16 from the hollow fibertangential flow filter 30 to the bioreactor vessel 11, to control thepressure within the hollow fiber tangential flow filter 30. For example,the valve 34 may be a non-invasive valve, which resides outside tubingcarrying the return flow 16 that squeezes the tubing to restrict andcontrol the flow, allowing the valve to regulate the applied pressure onthe membrane. Alternatively, or in addition, a flow controller 36 may beprovided at the pumphead 26 a inlet in order to provide pulsed flow tothe hollow fiber tangential flow filter 30, as described in more detailbelow. The permeate flow 24 may be continually removed from thebioreactor fluid 13 which flows through the hollow fiber tangential flowfilter 30. The pumphead 26 a and the permeate pump 22 are controlled bythe control system 20 to maintain the desired flow characteristicsthrough the hollow fiber tangential flow filter 30.

The pumphead 26 a and hollow fiber tangential flow filter 30 in thetangential flow filtering system 14 may be connected by flexible tubingallowing easy changing of the elements. Such tubing allows asepticreplacement of the hollow fiber tangential flow filter 30 in the eventthat the hollow fiber tangential flow filter 30 becomes plugged withmaterial and therefore provides easy exchange to a new hollow fiberassembly.

The tangential flow filtering system 14 may be sterilized, for example,using gamma irradiation, ebeam irradiation, or ETO gas treatment.

Referring again to FIG. 1, during operation, two permeate fluid outletports 32 b and 32 c may be employed to remove permeate flow 24 in insome embodiments. In other embodiments, only a single permeate fluidoutlet port may be employed. For example, permeate flow 24 may becollected from the upper permeate port 32 c only (e.g., by closingpermeate port 32 b) or may be collected from the lower permeate port 32b only (e.g., by draining the permeate flow 24 from the lower permeateport 32 b while the permeate port 32 c closed or kept open). In certainbeneficial embodiments, the permeate flow 24 may be drained from thelower permeate port 32 b to reduce or eliminate Sterling flow, which isa phenomenon where an upstream (lower) end of the of the hollow fibers60 (the high-pressure end) generates permeate that back-flushes thedownstream (upper) end of the hollow fibers 60 (the low-pressure end).Draining the permeate flow 24 from the lower permeate port 32 b leavesair in contact with the upper end of the of the hollow fibers 60minimizing or eliminating Sterling flow.

In certain embodiments, the bioreactor fluid 12 may be introduced intothe hollow fiber tangential flow filter 30 at a constant flow rate.

In certain embodiments, the bioreactor fluid may be introduced into thehollow fiber tangential flow filter 30 in a pulsatile fashion (i.e.,under pulsed flow conditions), which has been shown to increase permeaterate and volumetric throughput capacity. As used herein “pulsed flow” isa flow regime in which the flow rate of a fluid being pumped (e.g.,fluid entering the hollow fiber tangential flow filter) is periodicallypulsed (i.e., the flow has periodic peaks and troughs). In someembodiments, the flow rate may be pulsed at a frequency ranging from 1cycle per minute or less to 2000 cycles per minute or more (e.g.,ranging from 1 to 2 to 5 to 10 to 20 to 50 to 100 to 200 to 500 to 1000to 2000 cycles per minute) (i.e., ranging between any two of thepreceding values). In some embodiments, the flow rate associated withthe troughs is less than 90% of the flow rate associated with the peaks,less than 75% of the flow rate associated with the peaks, less than 50%of the flow rate associated with the peaks, less than 25% of the flowrate associated with the peaks, less than 10% of the flow rateassociated with the peaks, less than 5% of the flow rate associated withthe peaks, or even less than less than 1% of the flow rate associatedwith the peaks, including zero flow and periods of backflow between thepulses.

Pulsed flow may be generated by any suitable method. In someembodiments, pulsed flow may be generated using a pump such as aperistaltic pump that inherently produces pulsed flow. For example,tests have been run by applicant which show that switching from a pumpwith a magnetically levitated rotor like that described above underconstant flow conditions to a peristaltic pump (which provides a pulserate of about 200 cycles per minute) increases the amount of time that atangential flow depth filter can be operated before fouling (and thusincreases the quantity of permeate that can be collected).

In some embodiments, pulsed flow may be generated using pumps thatotherwise provide a constant or essentially constant output (e.g., apositive displacement pump, centrifugal pumps including magneticallylevitating pump, etc.) by employing a suitable flow controller tocontrol the flow rate. Examples of such flow controllers include thosehaving electrically controlled actuators (e.g. a servo valve or solenoidvalve), pneumatically controlled actuators or hydraulically controlledactuators to periodically restrict fluid entering or exiting the pump.For example, in certain embodiments, a flow controller 36 may be placedupstream (e.g., at the inlet) or downstream (e.g., at the outlet) of apump 26 like that described hereinabove (e.g., upstream of pumphead 26 ain FIG. 4A) and controlled by a controller 20 to provide pulsatile flowhaving the desired flow characteristics.

EXAMPLES

Tangential flow depth filters were tested which contained hollow fibershaving a lumen diameter of 1.5 mm and a wall thickness of 2.4 mm.However, other ranges of lumen diameters are contemplated throughoutthis disclosure. Hollow fibers having a mean pore size of 1 micron or 2microns were formed from bonded extruded bicomponent filaments having acore of polyethylene terephthalate and a sheath of polypropylene. Hollowfibers having a mean pore size of 0.5 micron, 1 micron, 2 microns or 4microns were also formed from bonded extruded bicomponent filamentshaving a core of polyethylene terephthalate and a sheath ofpolypropylene, which were subsequently provided with a coating ofpolyvinylidene fluoride (PVDF).

A fluid containing Chinese Hamster Ovary (CHO) cells was concentrated byrecycling the fluid though tangential flow depth filters containinghollow filters as described above using a peristaltic pump providing apulsatile flow at a pulse frequency of 200 cycles per minute. Runs wereconducted in concentration mode at 8000 s−1 shear rate (160 ml/min)using tangential flow depth filters having the following hollow fiberswith the following permeation flow rates, expressed as LMH (liters permeter2 per hour, or L/m2/h): (a) 1 micron noncoated hollow fiber, 300LMH, (b) 2 micron noncoated hollow fiber, 100 LMH, (c) 2 micron uncoatedhollow fiber, 300 LMH, (d) 2 micron coated hollow fiber, 100 LMH, and(e) 4 micron coated hollow fiber, 40 LMH increased to 100 LMH duringrun.

Results expressed as normalized permeate pressure versus time are shownin FIG. 5A. Final concentrations of cells in the retentate at the pointof filter fouling were as follows: 115.106 cells/ml (1 μm noncoated, 300LMH); 97.106 cells/ml (2 μm coated, 100 LMH,); 688.106 cells/ml (2noncoated, 300 LMH); 1.5.109 cells/ml (2 μm noncoated, 100 LMH); and72.106 cells/ml (4 μm coated, 40 and 100 LMH).

As seen from FIG. 5A, generally, pressure decay was quick at 300 LMH andfor the 4 μm fiber. 100 LMH appeared to be the most optimal forconcentration. Among the 2 μm fibers, the coated 2 μm fiber performedworse than the noncoated 2 μm fiber at 100 LMH. Each of the fibers ofFIG. 5A has a percent density. The 1 μm fiber has a percent density ofabout 55%, the 2 μm fiber has a percent density of about 53%, the 4 μmfilter has a density of about 51%. The 2 μm 53% density fibers performedbetter than both of the 1 μm 55% density and 4 μm 51% density fibers,with the 1 μm 55% density fiber performing the worst of these samples.Exemplary undesirable characteristics observed of these fibers includepassing too much turbidity and too many cells through the 4 μm 51%density fiber and a passage of fluid through the 1 μm 55% density at anundesirably rapid rate.

A fluid containing Chinese Hamster Ovary (CHO) cells was alsoconcentrated by initially pumping the fluid through the tangential flowdepth filter using a magnetically levitating pump having the followinghollow fibers at the following flow rates: 1 micron noncoated hollowfiber, 100 LMH and 2 micron coated hollow fiber, 100 LMH. Flow wasswitched from the magnetically levitating pump to a peristaltic pumpproviding a pulsatile flow at a pulse frequency of 200 cycles per minuteafter about 5 minutes for the 2 micron coated hollow fiber and afterabout 8 minutes for the 1 micron noncoated hollow fiber. Resultsexpressed as normalized permeate pressure versus time are shown in FIG.5B. As seen from FIG. 5B, normalized permeate pressure was negative forthe filters during the initial periods of operation using themagnetically levitating pump. After switching the peristaltic pump,however, the normalized permeate pressure turned positive, accompaniedby an increase in permeate flow.

While the disclosure herein disclosed has been described by means ofspecific embodiments and applications thereof, numerous modificationsand variations could be made thereto by those skilled in the art withoutdeparting from the scope of the disclosure set forth in the claims.

With reference to FIG. 6, data for VCD and percent viability over timefor an embodiment of a tangential flow filter for bioprocessingapplications is shown, which includes a procedure initiated while usinga first sintered filter P2 having a range of pore sizes of about 2 μm toabout 5 μm, followed by a second filter P3 replacing the first filterP2. The second filter P3 had pore sizes of about 4 μm and a densitypercentage of about 51%. The first sintered filter P2 fouled during aprocedure after about eight days of operation. During this eight-dayoperation, the first sintered filter P2 was unable to operate with morethan a VCD of about 60×106 cells/mL. After fouling, the first sinteredfilter P2 was replaced with the second filter P3. The second filter P3was exposed to more than 60×106 cells/mL for an operation period of ninedays with a 2 vessel volumes per day (VVD) exchange rate. A peak VCD ofthe system during operation with the second filter P3 was 175.0×106cells/mL. On the ninth day of the second filter's P3 operation(seventeenth day for the procedure overall) the permeate line of thesystem experienced a mechanical failure and began leaking. Therefore,the procedure was terminated. The second filter P3 maintained a largerVCD during its operation compared to the first sintered filter P2.

Table 1 below shows exemplary data of six filters having a densitypercentage of about 51%. Although the second filter P3 of FIG. 6 and thefilters of Table 1 below have a pore size of about 4 μm and a densitypercentage of about 51%, other filters are contemplated having adifferent pore size and density percentage, e.g., a filter having adensity percentage of about 53% and a pore size of about 2 μm with a 90%nominal retention.

TABLE 1 Parameters for Filters Having a Pore Size of About 4 μm Scale(sn B651486632) Caliper (SN 11344515) Sample Weight (g) Length (in) OD(cm) max OD (cm) min Avg ID (cm) Density 1 10.7 27.3 0.63246 0.629920.63119 0.15 0.522931121 2 13 33.46 0.64262 0.63246 0.63754 0.150.507494299 3 13 33.42 0.6477 0.63246 0.64008 0.15 0.503843298 4 5.814.88 0.64008 0.63246 0.63627 0.15 0.511296131 5 5.8 14.88 0.637540.62992 0.63373 0.15 0.515646644 6 5.9 14.88 0.635 0.63246 0.63373 0.150.524537103 Avg 0.514291433 StDev 0.00831614

With reference to FIG. 7, various metrics of a filter of FIG. 6 areshown. For example, an average percent of sieving through the secondfilter P3 is 99.24+14.85. Percent sieving refers to the volume of afluid that transfers through (e.g., across) a porous wall of a filter.The second filter's P3 peak VCD of 175.0×106 cells/mL is much higherthan that of the first sintered filter P2 of about 60×106 cells/mL,which was achieved without the second filter P3 fouling while the firstsintered filter P2 did foul.

With reference to FIG. 8, a cell growth profile of the second filter P3of FIGS. 6 and 7 is shown. The VVD range of the second filter P3 is 2. AVVD range and peak VCD of the second filter P3 is displayed in Table 2.

TABLE 2 VVD Range and Peak VCD of the Second Filter P3 VVD Range PeakVCD (106 cells/mL) P3 2 175.0

FIG. 9 and Table 3 show an average percent of sieving for the secondfilter P3 of FIGS. 6-8. The second filter P3 exhibited an averagepercent sieving of about 100% and maintained above about 80% throughoutits operation.

TABLE 3 Average Percent Sieving of the Second Filter P3 Avg % Sieving P399.24% ± 14.85%

FIG. 10 and Table 4 show a percent of cells passing through the secondfilter P3 of FIGS. 6-9. The initial percent of cells passing through thesecond filter P3 was initially much higher (about more than 1%) thanusual values observed in previous TFDF perfusion (e.g., less than about1%). The percent of cells passing decreased over the perfusion period ofthe second filter P3, with a spike in cells passing at the peak VCD.Cell Retention efficiency was maintained above about 95% throughoutperfusion culture.

TABLE 4 Passage of Cells Through the Second Filter P3 Peak % CellsPassing Peak VCD Passing Avg % Cells Passing Avg VCD Passing Avg CD (um)P3 4.79% 6.87 2.10% ± 1.49% 2.26 ± 1.42 10.13 ± 0.28

FIG. 11 and Table 5 show a flux of the second filter P3 run continuouslyusing a peristaltic pump of FIGS. 6-10, which is significantly linearacross the culture period.

TABLE 5 Flux of the Second Filter P3 VVD Range Flux Range (LMH) P3 224-39

FIG. 12 and Table 6 show a turbidity of the second filter P3 of FIGS.6-11. Turbidity values related to the second filter P3 were higher thanthe first filter P2.

TABLE 6 Turbidity of the Second Filter P3 Retentate Range (NTU) PermeateRange (NTU) P3 1720-625 354-1139

With reference to FIG. 13, transmembrane pressure (ATMP/sec) is observedat varying filter fluxes in TFDF systems utilizing 1.5 mm or 2.0 mm TDFinternal diameters. Significant increases in ATMP/sec are indicative ofthe formation of a gel layer on the inner surfaces of the tubularfiltration elements (in this case TDFs) and signal fouling of thefilter. The figure shows that, when operated at a fixed shear rate (y)of 8000 s−1, the 1.5 mm TFDF setup exhibited fouling at fluxes above 400L·m−2·hr−1, while the 2 mm TFDF setup exhibited no appreciable foulingat fluxes up to 2300 L·m−1 hr−1. Table 7, below, lists filter parametersand operating variables for both conditions; the systems differedprincipally in their respective TDF diameters and their Reynolds numbersat the feed, though different feed flow rates were used to achieve thesame shear rate in both systems.

TABLE 7 Filter Parameters and Operating Variables For 1.5 And 2 mm TFDFSystems 1.5 mm system 2 mm system TDF diameter (d) 1.5 mm 2.0 mmKinematic viscosity (μ) 1.0 cSt 1.0 cSt TDF cross-sectional area (A)1.767 mm² 3.142 mm² Feed flow rate (Q_(F)) $160\; \frac{mL}{\min}$$377\frac{mL}{\min}$ Feed Velocity (V_(F)) $1.509\frac{m}{s}$$2\frac{m}{s}$ Shear Rate (γ) 8048.131 s⁻¹ 8000.188 s⁻¹ Reynolds Numberat feed (Re_(F)) 2263.537 4000.094

CONCLUSION

The present disclosure is not limited to the particular embodimentsdescribed. The terminology used herein is for the purpose of describingparticular embodiments only, and is not intended to be limiting. Unlessotherwise defined, all technical terms used herein have the same meaningas commonly understood by one of ordinary skill in the art to which thedisclosure belongs.

Although embodiments of the present disclosure are described withspecific reference to cultured mediums, including for use inbioprocessing, it should be appreciated that such systems and methodsmay be used in a variety of configurations of processing fluids, with avariety of instruments, and a variety of fluids.

As used herein, the singular forms “a,” “an,” and “the” are intended toinclude the plural forms as well, unless the context clearly indicatesotherwise. The terms “comprises” and/or “comprising,” or “includes”and/or “including” when used herein, specify the presence of statedfeatures, regions, steps elements and/or components, but do not precludethe presence or addition of one or more other features, regions,integers, steps, operations, elements, components and/or groups thereof.As used herein, the conjunction “and” includes each of the structures,components, features, or the like, which are so conjoined, unless thecontext clearly indicates otherwise, and the conjunction “or” includesone or the others of the structures, components, features, or the like,which are so conjoined, singly and in any combination and number, unlessthe context clearly indicates otherwise. The term “or” is generallyemployed in its sense including “and/or” unless the content clearlydictates otherwise.

All numeric values are herein assumed to be modified by the term“about,” whether or not explicitly indicated. The term “about”, in thecontext of numeric values, generally refers to a range of numbers thatone of skill in the art would consider equivalent to the recited value(i.e., having the same function or result). In many instances, the term“about” may include numbers that are rounded to the nearest significantfigure. Other uses of the term “about” (i.e., in a context other thannumeric values) may be assumed to have their ordinary and customarydefinition(s), as understood from and consistent with the context of thespecification, unless otherwise specified. The recitation of numericalranges by endpoints includes all numbers within that range, includingthe endpoints (e.g. 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, and 5).

It is noted that references in the specification to “an embodiment”,“some embodiments”, “other embodiments”, etc., indicate that theembodiment(s) described may include a particular feature, structure, orcharacteristic, but every embodiment may not necessarily include theparticular feature, structure, or characteristic. Moreover, such phrasesare not necessarily referring to the same embodiment. Further, when aparticular feature, structure, or characteristic is described inconnection with an embodiment, it would be within the knowledge of oneskilled in the art to affect such feature, structure, or characteristicin connection with other embodiments, whether or not explicitlydescribed, unless clearly stated to the contrary. That is, the variousindividual elements described below, even if not explicitly shown in aparticular combination, are nevertheless contemplated as beingcombinable or arrangeable with each other to form other additionalembodiments or to complement and/or enrich the described embodiment(s),as would be understood by one of ordinary skill in the art.

What is claimed is:
 1. A bioprocessing system comprising: a bioreactor;a tangential flow depth filtration (TFDF) unit comprising a thick-walledhollow fiber formed from at least one polymer and comprising a porouswall having a pore size and a density, the porous wall defining a lumenthat is in fluid communication with the bioreactor; a permeate fluidoutlet in fluid communication with the porous wall; and a pump in fluidcommunication with the lumen; wherein the density is between 51% and 56%of the density of an equivalent solid volume of the polymer.
 2. Thebioprocessing system of claim 1, wherein the average pore size is about0.2 μm to about 10 μm with a 90% nominal retention.
 3. The bioprocessingsystem of claim 1, wherein the density is about 53%.
 4. Thebioprocessing system of claim 1, wherein the thick-walled hollow fiberis formed from melt-blown filaments of the polymer or sintered polymerparticles.
 5. The bioprocessing system of claim 1, wherein the porouswall has a thickness ranging from 2 mm to 7 mm.
 6. The bioprocessingsystem of claim 1, wherein the pump is configured to provide a pulsedflow of fluid through the lumen.
 7. A method of culturing cells in aperfusion bioreactor system comprising a culture vessel fluidlyconnected to a tangential flow depth filtration (TFDF) unit having aretentate channel and a filtrate channel, comprising the steps of:flowing a culture medium from the culture vessel through the retentatechannel of the TFDF unit, whereby a fraction of the culture mediumpasses into the filtrate channel; and returning fluid from the retentatechannel to the culture vessel; wherein (a) the culture medium comprisesat least 60×10⁶ cells/mL and (b) the method is performed for at least 8consecutive days.
 8. The method of claim 7, wherein at least 80% of thecells are viable throughout the 8 consecutive days.
 9. The method ofclaim 7, further comprising a step of adding to the system a volume offresh culture medium equal to a permeate volume.
 10. The method of claim9, wherein the step of adding the volume of fresh culture mediumcomprises adding at least 2 times a volume of the culture vessel to thesystem per day.
 11. The method of claim 7, wherein the culture mediumcomprises a bioproduct of interest, and a rate of sieving of thebioproduct of interest is at least 99% throughout the 8 consecutivedays.
 12. The method of claim 7, wherein the TFDF unit comprises aporous thick-walled hollow fiber comprising polymer, wherein a densityof the porous thick-walled hollow fiber is between 51% and 56% of thedensity of an equivalent solid volume of the polymer.
 13. The method ofclaim 12, wherein the density is about 53%.
 14. The method of claim 12,wherein a wall of the porous thick-walled hollow fiber has a thicknessof 0.2 mm to 7 mm.
 15. The method of claim 7, wherein the polymer isselected from the group consisting of polyolefin, a polyester, and acombination thereof.
 16. A method of harvesting a biomaterial from abioreactor system comprising a process vessel fluidly connected to atangential flow depth filtration (TFDF) unit having a feed/retentatechannel and a filtrate channel, comprising the steps of: pumping aculture medium comprising the biomaterial from the process vesselthrough the feed/retentate channel of the TFDF unit, whereby a fractionof the biomaterial passes into the filtrate channel; returning fluidfrom the feed/retentate channel to the process vessel; and collectingfluid from the filtrate channel; wherein the TFDF unit comprises athick-walled hollow fiber formed from a polymer and comprising a porouswall, the thick-walled hollow fiber having a density of about 53% of thedensity of an equivalent solid volume of the polymer, the porous walldefining a lumen of 3 mm to 7 mm that is in fluid communication with thefeed/retentate channel, the porous wall having a thickness of about 5mm; wherein the TFDF unit has a flux above about 400 L·m⁻²·hr⁻¹; whereinthe TFDF unit has a peak cell passage of under 5%; wherein a rate ofsieving of the bioproduct is at least 99%.
 17. A method of harvesting abio material from a bioreactor system comprising a process vesselfluidly connected to a tangential flow depth filtration (TFDF) unithaving a feed/retentate channel and a filtrate channel, comprising thesteps of: flowing a culture medium through the feed/retentate channel ofthe TFDF unit, whereby a fraction of the culture medium passes into thefiltrate channel; returning fluid from the feed/retentate channel to theprocess vessel; and collecting fluid from the filtrate channel; whereinthe TFDF unit comprises a thick-walled hollow fiber formed from at leastone polymer and comprising a porous wall, the thick-walled hollow fiberhaving a density of about 53% of the density of an equivalent solidvolume of the at least one polymer, the porous wall defining a lumenthat is in fluid communication with the feed/retentate channel.
 18. Themethod of claim 17, wherein a pore size of the porous wall is about 2 μmwith a 90% nominal retention.
 19. The method of claim 17, wherein theflowing step further comprises the use of a pump selected from the groupconsisting of a centrifugal levitating magnetic pump, a positivedisplacement pump, a peristaltic, a membrane pump, and an ATF pump. 20.The method of claim 17, wherein the hollow fiber comprises a lumenhaving an internal diameter of 0.75 mm to 5 mm therethrough.
 21. Themethod of claim 17, wherein the hollow fiber has a flux above about 40L·m⁻²·hr⁻¹.